Reversible thermally induced spin crossover in the myoglobin–nitrito adduct directly monitored by resonance Raman spectroscopy

Myoglobin has been demonstrated to function as a nitrite reductase to produce nitric oxide during hypoxia. One of the most intriguing aspects of the myoglobin/nitrite interactions revealed so far is the unusual O-binding mode of nitrite to the ferric heme iron, although conflicting data have been reported for the electronic structure of this complex also raising the possibility of linkage isomerism. In this work, we applied resonance Raman spectroscopy in a temperature-dependent approach to investigate the binding of nitrite to ferric myoglobin and the properties of the formed adduct from ambient to low temperatures (293 K to 153 K). At ambient temperature the high spin state of the ferric heme Fe–O–N 
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 O species is present and upon decreasing the temperature the low spin state is populated, demonstrating that a thermally-induced spin crossover phenomenon takes place analogous to what has been observed in many transition metal complexes. The observed spin crossover is fully reversible and is not due to linkage isomerism, since the O-binding mode is retained upon the spin transition. The role of the heme pocket environment in controlling the nitrite binding mode and spin transition is discussed.


Introduction
Heme-proteins achieve a wide and divergent range of physiological functions, with the prosthetic group of the Fe derivative of protoporphyrin IX being the vital key for their activities. [1][2][3] Among the most studied heme-proteins are vertebrate myoglobin (Mb) and hemoglobin (Hb), well-known for their roles in oxygen storage and transport. During the last few years, new functions of these proteins have emerged including their ability to function as nitrite reductases (NiRs) in mammals, transforming NO 2 − to NO and thus supporting NO signalling during metabolic stress and hypoxic conditions. [4][5][6][7] The reaction pathway involves NO 2 − binding to the heme Fe and thus, elucidating the structural properties and electronic conguration of various heme-nitrite adducts is crucial for understanding the biochemical reaction mechanism. Most studies on the reactions of NO 2 − with metalloproteins have been focusing on denitrication enzymes (Cu-NiRs and cytochrome cd 1 -NiRs) and more lately on the heme-globins, revealing interesting and diverse aspects of NO 2 − coordination and reduction chemistry. Nitrite is a bidentate ligand that can coordinate to metal centers in a bidentate motif as observed in Cu-NiRs, or via the N-atom (nitro-, N-binding mode) as in cytochrome cd 1 NiRs, or less commonly via one of its O-atoms (nitrito-, O-binding mode). [8][9][10][11][12][13][14] Linkage isomerism between the nitrito-and nitro-binding modes in metal complexes is possible and has been suggested to play important role in biochemical processes. [15][16][17][18][19] X-ray crystallographic studies revealed the unusual O-binding mode in ferric Mb and Hb, providing the rst examples of this coordination in metalloproteins and this mode was also retained upon photoreduction of the heme Fe in Mb. 11,12,14 However, EPR spectroscopy and DFT studies raised the possibility of nitrito/nitro linkage isomerism in solution for Mb and Hb. 20 Moreover, conicting data have been reported for the electronic conguration of the ferric heme Fe-nitrito (Fe-ONO) adduct. The ferric heme Fe-ONO adduct has been assigned to low spin (LS) or high spin (HS) or LS/HS equilibrium states by different research groups that employed various spectroscopic methods including cryo EPR, room temperature resonance Raman and MCD spectroscopies as well as theoretical calculations. [20][21][22][23][24][25] The spin state of heme complexes is closely related to their reactivity and thus important in dening the course of biochemical reactions. In fact, one should consider that ligand/ substrate binding to the heme Fe and/or electron transfer reactions are frequently accompanied by spin transitions that control heme-protein functions and mechanisms of enzymatic reactions. [1][2][3] In addition, external factors including temperature, light, pressure, electromagnetic elds as well as properties such as ligand type, coordination geometry and secondary bonding interactions can also give rise to spin crossover (SCO) phenomena in transition metal complexes and heme proteins. SCO phenomena are being studied in enzymes as they play vital roles in biochemical reactions, as well as in synthetic heme and non-heme systems due to their potential applications in nanotechnology, molecular electronics and spintronics. [26][27][28][29][30][31][32][33] Resonance Raman spectroscopy is a valuable tool for detection of SCO phenomena in heme proteins and we have previously utilized it to report an unusual ligand-concentration dependent spin transition in the green pigment of Mb, namely the ferric heme nitrito/2-nitrovinyl species. 23 The resonance Raman spectra of heme proteins comprise of multiple vibrational modes that are sensitive to oxidation, spin and coordination of the heme Fe center, while isotopic substitution of the ligand allows the observation of the binding mode to the heme Fe center. 23,34,35 In this work, we employed resonance Raman spectroscopy in a temperature-dependent approach to address if temperature can induce the formation of NO Raman data were collected by Horiba Scientic LabRAM spectrograph equipped with a CCD detector and an Olympus BX41 microscope. An Ondax SureLock LM-405 laser with an integrated CleanLine ASE lter was used to provide the excitation wavelength at 405 nm, and a 405 nm Semrock StopLine single-notch lter was used to reject the Rayleigh scattering. The samples were placed in temperature-controlled FTIR600 cell (Linkam Scientic Instruments). The desired temperature was achieved using the T95 and LN95 temperature controllers along with the use of liquid N 2 . The laser power incident on the sample was 20 mW and the total accumulation time for each measurement was 6-18 min. The Raman spectra were calibrated using toluene. OriginPro 2021 soware was used for spectra processing and analysis.

Results and discussion
The high-frequency region of the resonance Raman spectra of heme-proteins (1300-1700 cm −1 ) has several modes that are sensitive to the oxidation (n 4 ), coordination (n 3 ) and spin state (n 2 , n 10 ) of the heme Fe. The spectrum of metMb at ambient temperature includes vibrations that are characteristic of sixcoordinate HS (6cHS) heme at 1371 cm −1 (n 4 ), 1482 cm −1 (n 3 ) and 1563 cm −1 (n 2 ). 22,35 The resonance Raman spectrum of the reaction of metMb with NO 2 − at 293 K, shown in Fig. 1A, reveals a slight shi of the n 4 to 1373 cm −1 and n 2 to 1565 cm −1 , in agreement with previously published work in which we characterized the reaction species by resonance Raman spectroscopy and identied it as a 6cHS ferric heme Fe-ONO species at ambient temperature. 22,23 Fig. 1A also contains the resonance Raman spectra of the ferric Mb-nitrite adduct obtained by decreasing the temperature in 20 K steps, down to 153 K. Upon decreasing the temperature, the most readily observable change is the development of a band at 1646 cm −1 attributed to the n 10 of 6cLS species, indicating that a thermally-induced SCO transition occurs. 34 This is also conrmed by the decrease in the intensity of the n 2 of at 1565 cm −1 (6cHS) and concomitant increase of the 1586 cm −1 band that is attributed to the n 2 mode of 6cLS state. Similar observations are made for the HS and LS components of the n 3 mode at 1482 cm −1 and 1512 cm −1 , respectively. Moreover, upshi of the n 4 band is observed as temperature decreases, shiing from 1373 cm −1 at 293 K to 1377 cm −1 at 153 K. An enlarged view of the n 4 mode is shown in Fig. 1B, demonstrating the presence of the components at 1373 cm −1 and 1377 cm −1 . Overall, the 6cHS species is detected at room temperature, while the 6cLS species is progressively populated when temperature decreases and even at 153 K the 6cHS species is present along with the 6cLS complex. It is noted that the SCO transition we observe is fully reversible and the 6cLS species is reconverted to the 6cHS state when temperature increases to 293 K. The transition temperature for the spin crossover, T 1/2 = 230 K, was calculated by plotting the 1646 cm −1 peak area (n 10 of the 6cLS species) versus temperature and tting of the data, as shown in Fig. S1 (ESI †). The next important question to address is if the thermallyinduced SCO transition we observe could be due to linkage isomerism. Linkage isomerism, that is the presence of both nitrito and nitro-binding modes which can easily interconvert, was previously suggested by cryo EPR experiments and DFT calculations for Mb and Hb. 20 The nitrite ion, when N-bonded, is a strong eld ligand yielding a 6cLS heme species. Resonance Raman spectroscopy can discriminate between the nitro-and nitrito-binding modes by identication of the substrate-bound vibrations. 22,23,36,37 We have previously demonstrated that at ambient temperature the ferric HS heme Fe-O-N]O Mb adduct is formed in solution and reported its detailed vibration characterization by detecting and assigning the n(Fe-ONO), d(FeONO), n(N-O) and n(N]O) vibrational frequencies. 23 Herein, we attempt to characterize the corresponding equilibrium HS/LS species that we detect at low temperature. Fig. 2 shows the high-frequency region of the resonance Raman spectrum of metMb (trace a) and aer its reaction with 14    feature that was not previously observed in the ambient temperature low-frequency resonance Raman data is the mode at 926 cm −1 that shis to 909 cm −1 in the 15 36,37 The fact that we have not been able to identify distinct bands for the n(Fe-ONO) and d(FeONO) of the LS heme Fe-ONO species may indicate that these fall within the same range of the HS adduct or are not observed under the 405 nm excitation.
The observed lower frequency of the n(N]O) and higher frequency of the n(N-O) of the LS heme Fe-ONO compared to the HS heme Fe-ONO species indicate that different hydrogen bonding interactions and/or geometries of the FeONO moiety can exist for the two populations. Evidence for the importance of H-bonding interactions on the NO 2 − coordination mode and geometry have been provided by the crystallographic, spectroscopic and theoretical studies. [11][12][13]21,22,24,40 The crystal structure of the ferric Mb-nitrito adduct revealed that the FeONO moiety was in the trans conformation and the distal residue His64 was proposed to exert a strong inuence in directing the O-binding mode by H-bonding to O1 (Fe-O1-N]O2) as shown in Fig. 4, a hypothesis that was further supported with the observation of the N-binding mode in the crystal structure of the H64V Mb mutant. 11,13 Moreover, reintroduction of a H-bonding residue into the distal pocket in the H64V/V67R double mutant restored the O-binding mode of nitrite, but in a distorted cis-like conformation. 13 In the case of the nitrite adduct of human tetrameric Hb that also exhibits O-binding, the FeONO moiety was found in the trans conformation in the a subunit, while   24 Interestingly, second sphere control of spin state through Hbonding has been recently described in model ferric porphyrin complexes. Examples include ve coordinate Fe(III) octaethyltetraarylporphyrin, Fe(III) porphyrin complexes with covalently attached imidazole or thiolate ligands and axial H 2 O or OH − ligands and heme-peroxo-copper complexes. [30][31][32] In the latter study, intramolecular H-bonding interactions were demonstrated to facilitate thermally induced SCO in the synthetic heme-peroxo-copper complexes. 32 In the case of the temperature-induced SCO of the heme Fe-ONO reported here, the data are consistent with subtle conformational changes in the distal site affecting the hydrogen bonding interactions and/ or geometry of the FeONO moiety, although contributions from proximal effects should also be considered. We have previously utilized resonance Raman spectroscopy at ambient temperature to investigate a ligand-concentration dependent spin transition in ferric heme nitrito/2-nitrovinyl species. We suggested that structural rearrangements in the protein binding pocket accompanied by a change in the displacement of the heme Fe along the heme plane without breaking of the heme-ligand bonds were responsible for the spin state transition. 23 Conformational changes to the E helix in the distal environment, along with the F helix in the proximal site upon removal of excess ligand from the protein cavities were considered as regulating factors. 23,24 The role of the proximal environment in spin transitions is supported by DFT calculations that showed decrease of the Fe-N His bond length by ∼0.3 Å in the LS state compared to the HS species, while the corresponding Fe-O was less affected. 24 Bond contraction was also implicated in the temperature induced SCO in neuronal nitric oxide synthase bound with heme-coordinating thioether inhibitors. It was proposed that contraction of the Fe-S thioether bond by ∼0.2 Å below 200 K resulted in an increase in the ligand eld strength forcing the pairing of the iron d electrons into the lower energy orbitals and leading to a HS to LS transition. 27 Finally, we note that the HS state of the heme Fe-ONO species is dominant at ambient temperature and present even at 153 K along with the LS state. The temperature-dependent spectral changes that we observe in the resonance Raman experiments are gradual and reversible, thus indicating that the HS and LS states are in thermal equilibrium. Even though the theoretical studies predict that the energy differences between nitro-and nitrito-isomers are very low for ferric and ferrous porphyrins and thus isomers could interconvert by temperature variations, [19][20][21]41 this is not the case for the ferric nitrito-Mb. The protein environment controls the O-binding mode and the thermally induced spin transition is not due to linkage isomerism. Assuming that the O-binding mode is retained upon heme reduction, there would be the advantage of the requirement of a single proton transfer to lead to the formation and release of NO during the catalytic cycle. 41

Conclusions
In summary, the present work reports the direct observation of a thermally induced SCO in ferric nitrito-Mb by the application of resonance Raman spectroscopy in a temperature-dependent approach. The observed SCO is not the result of linkage isomerism, since the O-binding mode is retained upon the reversible HS to LS transition. The SCO behaviour of nitrito-Mb underscores the role of the heme pocket in not only orienting ligands/ substrates to allow selective reactions, but also in controlling properties such as the spin state that ultimately dene reactivity.

Conflicts of interest
There are no conicts to declare.